The objective of the proposed research is to investigate the mechanism of action of collagenases, which are unique in their ability to catalyze the degradation of collagen under physiological conditions. Collagen, the major structural protein of connective tissue, is highly resistant to attack by all known digestive and intracellular enzymes. Hence, the action of collagenases is essential to all processes involving the degradation or remodelling of connective tissue. Collagenases, which are believed to be zinc metalloenzymes, will be isolated from various sources according to published procedures and by affinity chromatographic methods to be developed. New and rapid fluorometric assay procedures will be developed to expedite the isolation and purification steps. The enzymes obtained will be characterized with respect to their homogeneity, metal content, amino acid composition, molecular weight and subunit structure. The role of the metal atom(s) in catalysis will be investigated by examining the effect of metal substitutions on enzymatic activity. Chemical modification reactions will be carried out to identify essential amino acid residues. The specificity of the enzyme toward synthetic substrates will be examined and the results used to design potent, specific inhibitors. This research should promote a greater understanding of the role of collagenases in mammalian connective tissue metabolism.